Consumable Cryopreserved Cells Transiently Overexpressing Gene(s) Encoding Drug Transporter Protein(s) and/or Drug Metabolizing Enzyme(s)

ABSTRACT

The present invention discloses cryopreserved recombinant cells for screening drug candidates that transiently overexpress one or more drug transporter proteins and/or drug metabolizing enzymes. Advantageously, such cells provide a cost-efficient consumable product that streamlines the process of screening whether drug candidates are substrates or inhibitors of drug transporter proteins and/or drug metabolizing enzymes.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is filed under 35 U.S.C. §111 as a continuationapplication of U.S. application Ser. No. 15/163,218, filed on May 24,2016, which is a continuation of U.S. application Ser. No. 14/972,012,filed on Dec. 16, 2015, which is a division of U.S. application Ser. No.14/644,000, filed on Mar. 10, 2015, which is a continuation applicationof International Application No. PCT/US2013/059152, filed on Sep. 11,2013, which designates the United States and claims priority to U.S.Provisional Patent Application No. 61/699,466, filed on Sep. 11, 2012,the entire contents of which are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to cryopreserved recombinant cellstransiently overexpressing one or more genes encoding drug transporterprotein and/or drug metabolizing enzyme such that activity of theencoded protein(s) is detectable in a population of said cells followingthaw from cryopreservation.

BACKGROUND OF THE INVENTION

Drug development is a costly and time consuming endeavor whereby drugcandidates must satisfy certain criteria established by governmentagencies such as the U.S. Food and Drug Administration and EuropeanMedicines Agency prior to receiving regulatory approval for marketingthereof. Importantly, assays are conducted to screen drug candidates todetermine whether any are substrates or inhibitors of one or more drugtransporter proteins and/or drug metabolizing enzymes as that can have asignificant effect on the absorption, distribution, metabolism andelimination of such drugs, their toxicity and drug-drug interactions.

Although cell lines stably expressing a gene encoding a drug transporterproteins or a drug metabolizing enzyme may be used for such screening,significant time and resources are required to generate and maintainfrozen stocks thereof. Plus, the level of recombinant protein expressedis typically variable (laboratory to laboratory) and may deteriorateand/or become more variable over time with passage of such cells.Alternatively, freshly plated cells either stably or transientlyexpressing a gene encoding a drug transporter protein or a drugmetabolizing enzyme may be employed for such screening. However, freshlyplated cells have a limited shelf life of a few days and are difficultto ship in a manner that maintains their viability. In addition, togenerate stable cell lines, the foreign transfected gene is actuallyintegrated into the host genome of the cells and carried along with itduring cycles of cells division. The chromosomal integration in the hostcells will lead to permanent modification of host genome, potentiallyleading to abnormal expression of other genes causing unexpected changesof host behavior and unreliable experimental results. Thus, there is aneed for cells suitable for screening drug candidates that reduces theinvestment of time and resources associated with drug development andprovide reliable results.

SUMMARY OF THE INVENTION

The present invention provides cryopreserved recombinant cells suitablefor screening drug candidates to determine whether any are substrates orinhibitors of one or more drug transporter proteins and/or drugmetabolizing enzymes that provide reliable results and readyconvenience. Desirably, the level of activity in a population of therecombinant cells following cryopreservation is comparable to that offreshly transfected cells. Additionally, the cryopreserved recombinantcells are readily packaged in a vial and shipped with dry ice or dryshipper and conveniently stored at −135° C. in liquid nitrogen uponreceipt with several years shelf life. Hence, such cells provide the enduser a consumable “thaw and use” product which provides convenience inconducting experiments and reduces the investment of time and resourcesin creating and/or maintaining cell stocks for screening drugcandidates.

In one aspect, the present invention provides cryopreserved recombinantcells including one or more transiently overexpressed genes encoding aprotein selected from the group consisting of a drug transporter proteinand a drug metabolizing enzyme, or a combination thereof whereinactivity of the drug transporter protein or the drug metabolizing enzymeor combinations is detectable in a population of the cryopreservedrecombinant cells following thaw from cryopreservation.

In another aspect, the present invention provides processes of preparingtransiently transfected recombinant cells which transientlyoverexpresses one or more genes encoding a protein selected from a drugtransporter protein and a drug metabolizing enzyme or a combinationthereof including transiently transfecting cells with one or more genesencoding a drug transporter protein or a drug metabolizing enzyme andcryopreserving the transiently transfected recombinant cells within 48hrs of transfection.

These and other features of the invention will be better understoodthrough a study of the following detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph of the percentage of viable cells from cell stock,cells after electroporation (EP) and cells after thaw fromcryopreservation for FreeStyle™ 293-F (FS293) cells and 293-F cellsgrown in suspension.

FIG. 2 are images of transfected cells 4 hrs (A), 24 hrs (B) and 48 hrs(C) after plating following thaw from cryopreservation.

FIG. 3 are images of 293-F cells transfected with pOATP1B1 expressionplasmid plated at (A) 0.4×10⁶ viable cells per well and (B) 0.2×10⁶viable cells per well in 24-well poly-D-lysine coated plates andcultured in plating media at 24 hrs post-plating (following thaw fromcryopreservation).

FIG. 4 are images of 293-F cells transfected with MATE1, MATE2K OATP1B3,long isoform OAT1 (full length cDNA with 563 amino acids; referred toherein as “OAT1 long”), short isoform OAT1 (missing 13 amino acid atC-terminus 522-534, with 550 amino acids; referred to herein as “OAT1short”), OAT3, and pCMV vector plated at 0.4×10⁶ cells per well in24-well poly-D-lysine coated plates at 24 hrs post-plating (followingthaw from cryopreservation).

FIG. 5 are fluorescence images of adhered HEK293 cells transfected with50 μg/ml, 100 μg/ml or 200 μg/ml green fluorescent protein (GFP) 24 hrs(A) and 48 hrs (B) following EP.

FIG. 6 is a graph of the percentage of viable cells following EP ofadhered HEK293 cells using varying amounts of DNA.

FIG. 7A is a graph of estradiol-17β-glucuronide (E17βG) uptake activityfollowing various incubation times in adhered HEK293 cells transfectedwith varying amounts of DNA (i.e., 0, 50 μg/ml, 100 μg/ml, 200 μg/ml or400 μg/ml OATP2/OATP1B1) at 48 hrs post EP.

FIG. 7B is a graph of estradiol-17β-glucuronide (E17βG) uptake activityfollowing various incubation times in adhered HEK293 cells transfectedwith varying amounts of DNA (i.e., 0, 50 μg/ml, 100 μg/ml, 200 μg/ml or400 μg/ml OATP2/OATP1B1) at 96 hrs post EP.

FIG. 8 is a graph of signal to noise ratio of estradiol-17D-glucuronide(E17βG) uptake following various incubation times in adhered HEK293cells transfected with varying amounts of DNA (i.e., 0, 50 μg/ml, 100μg/ml, 200 μg/ml or 400 μg/ml OATP2/OATP1B1) at 48 hrs post EP.

FIG. 9 is a graph of estradiol-17β-glucuronide (E17βG) uptake activityin adhered HEK293 cells transfected with either OATP2/OATP1B1 using asmall scale EP device (OC400), OATP2/OATP1B1 using a large scale EPdevice (CL2), or an empty vector control.

FIG. 10 is a graph of signal to noise ratio of estradiol-17D-glucuronide(E17βG) uptake following various incubation times in adhered HEK293cells transfected with OATP1B1 gene using either “Control” (i.e.,traditional lipid transfection reagent (lipofectamine 2000, availablefrom Invitrogen)) or STX, MaxCyte scalable EP device.

FIG. 11 is a graph of signal to noise ratio of estradiol-17D-glucuronide(E17βG) uptake following various incubation times in adhered HEK293cells transfected with OATP1B1 that are freshly plated or platedfollowing thaw from cryopreservation.

FIG. 12 are images of HEK293 cells transfected with OATP1B1*1a (GeneAccession No. NM_006446.4), OATP1B1*1b (Gene Accession No. NM_006446.3),OATP1B3, pCMV vector, long isoform OAT1 (full length cDNA with 563 aminoacids), OAT3, OCT1 or OCT2 using MaxCyte scalable EP device and scale-upprocess followed by cryopreservation, thawing, plating on Poly-D-Lysineplates and incubation for 24 hrs post-plating.

FIG. 13A is a graph depicting results of a time-dependent assay ofp-Aminohippuric acid (PAH) (prototypical substrate for OAT1) uptake inHEK293 cells overexpressing OAT1 or pCMV vector following variousincubation times (i.e., 1, 2, 5, 10 and 15 min.) with PAH at aconcentration of 3 μM.

FIG. 13B is a graph depicting results of a kinetic assay whereby uptakeof PAH at a concentration in the range of 3 to 200 μM was measured inHEK293 cells overexpressing OAT1 following incubation for 5 min. Km andVmax, calculated using Sigma-plot, are shown as insert in the graph.

FIG. 13C is a graph depicting results of an inhibition assay wherebyHEK293 cells overexpressing OAT1 were incubated with PAH at aconcentration of 15 μM and probenecid (OAT1 inhibitor) at aconcentration in the range of 0-300 μM for 5 min. IC50, calculated usingSigma-plot, is shown as insert in the graph.

FIG. 14A is a graph depicting results of a time-dependent assay ofEstrone-3-sulfate (E3S) (prototypical substrate for OAT3) uptake inHEK293 cells overexpressing OAT3 or pCMV vector following variousincubation times (i.e., 1, 2, 5, 10 and 15 min.) with E3S at aconcentration of 1 μM.

FIG. 14B is a graph depicting results of a kinetic assay whereby uptakeof E3S at a concentration in the range of 0.5 to 32 μM was measured inHEK293 cells overexpressing OAT3 following incubation for 1 min. Km andVmax, calculated using Sigma-plot, are shown as insert in the graph.

FIG. 14C is a graph depicting results of an inhibition assay wherebyHEK293 cells overexpressing OAT3 were incubated with E3S at aconcentration of 4 μM and probenecid (OAT3 inhibitor) at a concentrationin the range of 0-300 μM for 5 min. IC50, calculated using Sigma-plot,is shown as insert in the graph.

FIG. 15A is a graph depicting results of a time-dependent assay of TEA(prototypical substrate for OCT1) uptake in HEK293 cells overexpressingOCT1 or pCMV vector following various incubation times (i.e., 1, 2, 5,10 and 15 min.) with TEA at a concentration of 31 μM.

FIG. 15B is a graph depicting results of a time-dependent assay ofmetformin (prototypical substrate for OCT 1) uptake in HEK293 cellsoverexpressing OCT1 or pCMV vector following various incubation times(i.e., 1, 2, 5, 10 and 15 min.) with metformin at a concentration of 3.8μM.

FIG. 15C is a graph depicting results of a concentration-dependent assaywhereby uptake of TEA at a concentration of 1, 10 and 100 μM wasmeasured in HEK293 cells overexpressing OCT1 or pCMV vector followingincubation for 10 min.

FIG. 15D is a graph depicting results of a concentration-dependent assaywhereby uptake of metformin at a concentration of 0.1, 1 and 10 μM wasmeasured in HEK293 cells overexpressing OCT1 or pCMV vector followingincubation for 10 min.

FIG. 15E is a graph depicting results of an inhibition assay wherebyHEK293 cells overexpressing OCT1 were incubated with TEA and OCT1inhibitor (quinidine, verapamil or decynium-22) at variousconcentrations in the range of 0.1-500 μM for 10 min.

FIG. 15F is a graph depicting results of an inhibition assay wherebyHEK293 cells overexpressing OCT1 were incubated with metformin at aconcentration of 3.8 μM and OCT1 inhibitor cimetidine at variousconcentrations in the range of 4 μM to 3 mM for 10 min.

FIG. 16A is a graph depicting results of a time-dependent assay of TEA(prototypical substrate for OCT2) uptake in HEK293 cells overexpressingOCT2 or pCMV vector following various incubation times (i.e., 1, 2, 5,10 and 15 min.) with TEA at a concentration of 31 μM.

FIG. 16B is a graph depicting results of a time-dependent assay ofmetformin (prototypical substrate for OCT2) uptake in HEK293 cellsoverexpressing OCT2 or pCMV vector following various incubation times(i.e., 1, 2, 5, 10 and 15 min.) with metformin at a concentration of 3.8μM.

FIG. 16C is a graph depicting results of a concentration-dependent assaywhereby uptake of TEA at a concentration of 1, 10 and 100 μM wasmeasured in HEK293 cells overexpressing OCT2 or pCMV vector followingincubation for 10 min.

FIG. 16D is a graph depicting results of a concentration-dependent assaywhereby uptake of metformin at a concentration of 0.1, 1 and 10 μM wasmeasured in HEK293 cells overexpressing OCT2 or pCMV vector followingincubation for 10 min.

FIG. 16E is a graph depicting results of an inhibition assay wherebyHEK293 cells overexpressing OCT2 were incubated with metformin at aconcentration of 3.8 μM and OCT2 inhibitor cimetidine at a concentrationin the range of 4 μM to 3 mM for 10 min. IC50, calculated usingSigma-plot, is shown as insert in the graph.

FIG. 17A is a graph depicting results of a time-dependent assay ofestradiol-17β-glucuronide (E17βG) uptake in HEK293 cells overexpressingOATP1B1*1a or pCMV vector following various incubation times (i.e., 1,2, 5, 10 and 15 min.) with E17βG at a concentration of 1 μM.

FIG. 17B is a graph depicting results of a time-dependent assay ofestrone-3-sulfate (E3S) uptake in HEK293 cells overexpressing OATP1B1*1aor pCMV vector following various incubation times (i.e., 1, 2, 5, 10 and15 min.) with E3S at a concentration of 1 μM.

FIG. 17C is a graph depicting results of a time-dependent assay ofrosuvastatin uptake in HEK293 cells overexpressing OATP1B1*1a or pCMVvector following various incubation times (i.e., 1, 2, 5, 10 and 15min.) with rosuvastatin at a concentration of 1 μM.

FIG. 17D is a graph depicting results of a concentration-dependent assaywhereby uptake of E17βG at a concentration in the range of 0.25 to 40 μMwas measured in HEK293 cells overexpressing OATP1B1*1a followingincubation for 1 min. Km and Vmax, calculated using Sigma-plot, areshown as insert in the graph.

FIG. 17E is a graph depicting results of a concentration-dependent assaywhereby uptake of rosuvastatin at a concentration in the range of 0.78to 50 μM was measured in HEK293 cells overexpressing OATP1B1*1afollowing incubation for 5 min. Km and Vmax, calculated usingSigma-plot, are shown as insert in the graph.

FIG. 17F is a graph depicting results of an inhibition assay wherebyuptake of E17βG at a concentration of 1 μM was measured in HEK293 cellsoverexpressing OATP1B1*1a following incubation with inhibitorcyclosporin A at a concentration in the range of 0.04 to 30 μM for 5min. IC50, calculated using Sigma-plot, is shown as insert in the graph.

FIG. 18A is a graph depicting results of a time-dependent assay of E17βGuptake in HEK293 cells overexpressing OATP1B1*1b or pCMV vectorfollowing various incubation times (i.e., 1, 2, 5, 10 and 15 min.) withE17βG at a concentration of 1 μM.

FIG. 18B is a graph depicting results of a time-dependent assay of E3Suptake in HEK293 cells overexpressing OATP1B1*1b or pCMV vectorfollowing various incubation times (i.e., 1, 2, 5, 10 and 15 min.) withE3S at a concentration of 1 μM.

FIG. 18C is a graph depicting results of a time-dependent assay ofrosuvastatin uptake in HEK293 cells overexpressing OATP1B1*1b or pCMVvector following various incubation times (i.e., 1, 2, 5, 10 and 15min.) with rosuvastatin at a concentration of 1 μM.

FIG. 18D is a graph depicting results of a concentration-dependent assaywhereby uptake of E17βG at a concentration in the range of 0.25 to 40 μMwas measured in HEK293 cells overexpressing OATP1B1*1b followingincubation for 1 min. Km and Vmax, calculated using Sigma-plot, areshown as insert in the graph.

FIG. 18E is a graph depicting results of an inhibition assay wherebyuptake of E17βG at a concentration of 1 μM was measured in HEK293 cellsoverexpressing OATP1B1*1b following incubation with inhibitorcyclosporin A at a concentration in the range of 0.04 to 30 μM for 5min. IC50, calculated using Sigma-plot, is shown as insert in the graph.

FIG. 19A is a graph depicting results of a time-dependent assay ofcholecystokinin (CCK-8) uptake in HEK293 cells overexpressing OATP1B3 orpCMV vector following various incubation times (i.e., 1, 2, 5, 10 and 15min.) with CCK-8 at a concentration of 1 μM.

FIG. 19B is a graph depicting results of a time-dependent assay of E17βGuptake in HEK293 cells overexpressing OATP1B3 or pCMV vector followingvarious incubation times (i.e., 1, 2, 5, 10 and 15 min.) with E17βG at aconcentration of 1 μM.

FIG. 19C is a graph depicting results of a concentration-dependent assaywhereby uptake of CCK-8 at a concentration in the range of 0.5 to 20 μMwas measured in HEK293 cells overexpressing OATP1B3 following incubationfor 1 min. Km and Vmax, calculated using Sigma-plot, are shown as insertin the graph.

FIG. 19D is a graph depicting results of a concentration-dependent assaywhereby uptake of rosuvastatin at a concentration in the range of 0.78to 50 μM was measured in HEK293 cells overexpressing OATP1B3 followingincubation for 5 min. Km and Vmax, calculated using Sigma-plot, areshown as insert in the graph.

FIG. 19E is a graph depicting results of an inhibition assay wherebyuptake of CCK-8 at a concentration of 1 μM was measured in HEK293 cellsoverexpressing OATP1B3 following incubation with inhibitor cyclosporin Aat a concentration in the range of 0.04 to 30 μM for 2 min. IC50,calculated using Sigma-plot, is shown as insert in the graph.

DETAILED DESCRIPTION OF THE INVENTION

As used herein the following terms shall have the definitions set forthbelow.

As used herein, the term “cell” includes both primary cells as well asestablished cell lines (e.g., human embryonic kidney HEK293 cells,Chinese hamster ovary CHO, Madin-Darby Canine Kidney Cells MDCK, PigKidney Epithelial Cells LLC-PK1, human epithelial colorectaladenocarcinoma cells Caco-2 and Chinese hamster lung fibroblast V79cells).

As used herein, the term “drug transporter protein” refers to a membranebound transport protein that includes, but is not limited to ATP bindingcassette (ABC) transporters and solute carrier (SLC) transporters.

As used herein, the term “drug metabolizing enzyme” includes, but is notlimited to, cytochromes such as cytochrome (CYP) P450; UDP-glucouronyltransferase and other non-CYP drug metabolizing enzymes such as alcoholdehydrogenase, monoamine oxidase and aldehyde oxidase.

As used herein, the term “detectable” means that the activity of aselected probe substrate in cells transfected with a drug transporterprotein and/or drug metabolizing enzyme shall be higher than theactivity of the same probe substrate in cells transfected with emptyvector; desirably, the difference in activity will be at least 5-fold.

As used herein, the use of upper case letters in transporternomenclature identifies the human protein/gene, i.e., MRP2/ABCC2, etc.;smaller case letters indicate the transporter derives from a preclinical(nonhuman mammalian) species, i.e., Mrp2/Abcc2, etc. Unless otherwisespecified, a gene is derived from any species (e.g., human or othermammal).

As used herein, the terms “OATP1B1” “OATP2” and “SLCO1B1” areinterchangeable and refer to a human protein/gene that corresponds tothe nonhuman protein/gene Oatp2. Unless noted otherwise, reference toOATP1B1 is to OATP1B1*1b.

As used herein, the terms “OAT1” and “SLC22A6” are interchangeable andrefer to an organic anion transporter 1. Unless noted otherwise,reference to OAT1 is to the full length cDNA encoding with 563 aminoacids (also referred to herein as “OAT1 long”).

Exemplary ABC transporters include, but are not limited to those listedbelow in Table 1.

TABLE 1 GENE NAME PROTEIN NAME MDR1/P-gp/ABCB1 Multidrug ResistanceProtein 1 MRP1/ABCC1 Multidrug resistance protein 1 MRP2/ABCC2 Multidrugresistance-associated protein 2 MRP3/ABCC3 Multidrug resistance protein3 MRP4/ABCC4 Multidrug resistance protein 4 MRP5/ABCC5 Multidrugresistance protein 5 MRP6/ABCC6 Multidrug resistance protein 6MRP7/ABCC7 Multidrug resistance protein 7 MRP8/ABCC8 Multidrugresistance protein 8 BCRP/ABCG2 Breast Cancer Resistance ProteinBSEP/ABCB11 Bile Salt Export Pump

Exemplary SLC transporters include, but are not limited to those listedbelow in Table 2.

TABLE 2 GENE NAME PROTEIN NAME OSTα Organic solute transporter α OSTβOrganic solute transporter β OATP1B1*/SLCO Organic anion-transportingpolypeptide 1B1 1B1/OATP2 OATP1B3/SLCO 1B3 Organic anion-transportingpolypeptide 1B3 OAT1/SLC22A6 Organic anion transporter 1 OAT2/SLC22A7Organic anion transporter 2 OAT3/SLC22A8 Organic anion transporter 3OAT4/SLC22A11 Organic anion transporter 4 OCT1/SLC22A1 Organic cationtransporter 1 OCT2/SLC22A2 Organic cation transporter 2 OCT3/SLC22A3Organic cation transporter 3 OATP1A2/SLCO1A2 Organic anion-transportingpolypeptide 1A2 OATP2B1/SLCO2B1 Organic anion-transporting polypeptide2B1 PEPT1/SLC15A1 Peptide Transporter 1 PEPT2/SLC15A2 PeptideTransporter 2 OCTN1/SLC22A4 Organic cation/ergothioneine transporterOCTN2/SLC22A5 Organic cation/carnitine transporter MATE1/SLC47A1Multidrug and toxin extrusion 1 MATE2K/SLC47A2 Multidrug and toxinextrusion 2K URAT1/SLC22A12 Urate Transporter 1 ASBT/SLC10A2 Apicalsodium/bile acid co-transporter NTCP/SLC10A1 Sodium/taurocholateco-transporting peptide *In this instance, OATP1B1 includes OATP1B1*1aand OATP1B1*1b.

Exemplary SLC transporters tested, but are not limited to those listedbelow in Table 3.

TABLE 3 GENE ACCESSION GENE NAME FULL NAME NUMBER OATP1B1*1a/ Organicanion-transporting NM_006446.4 SLCO1B1*1a polypeptide 1B1 Wild Type(388A) OATP1B1*1b/ Organic anion-transporting NM_006446.3 SLCO1B1*1bpolypeptide 1B1 SNP 388A > G OATP1B3/ Organic anion-transportingNM_019844 SLCO1B3 polypeptide 1B3 OAT1/SLC22A6 Organic anion transporter1 NM_004790 OAT3/SLC22A8 Organic anion transporter 3 NM_004254OCT1/SLC22A1 Organic cation transporter 1 NM_003057 OCT2/SLC22A2 Organiccation transporter 2 NM_003058

Cells suitable for use in the present invention include mammalian cells,for example, as derived from human or non-human (e.g., mouse, rat, dog,monkey, hamster and pig, etc.). In certain embodiments, the cells arehepatocytes, or endothelial cells.

Gene delivery systems for introducing gene(s) into a population of cellsare known to a skilled artisan. Virus-based gene delivery methods may beused but require special handling of the cells due to safety concerns.Although lipid-based transfection methods may be used, lipid-basedtransfection reagents are relatively costly and such methods are notamenable to large-scale manufacturing processes. Additionally,lipid-based transfection methods result in relatively low gene deliveryefficiency and relatively delayed protein expression (generally 72 to 96hours post transfection) (data not shown). Electroporation (EP) ispreferable as it is amenable to large-scale manufacturing processes andavoids the safety issues of viral-based gene delivery methods. Further,EP results in relatively efficient gene delivery.

After gene delivery into a population of cells, gene(s) encoding a drugtransporter protein and/or a drug metabolizing enzyme will beoverexpressed such that activity of the protein(s) encoded therefrom aredetectable following thaw from cryopreservation. Drug candidates can betested to determine if any are substrates or inhibitors of theprotein(s) encoded from the overexpressed gene(s) by incubation of therecombinant cells therewith. In particular, if a drug candidate is asubstrate of a drug transporter protein and/or a drug metabolizingenzyme, the drug candidate will be affected. For instance, if the drugcandidate is a substrate of a drug transporter protein, the drugcandidate will be translocated in or out of the recombinant cell via thedrug transporter protein. However, if the drug candidate is an inhibitorof the drug transporter protein, the drug candidate will inhibittranslocation of a substrate of the drug transporter protein in or outof the recombinant cell.

Alternatively, assays can be conducted using whole cells or subcellularfractions thereof (microsome/cytosol).

Additionally, recombinant cells of the present invention may be furthertransfected with RNAi or siRNA of the transiently overexpressed gene(s)to knockdown/knockout the expression thereof as is desirable for certainassays. Primary cells (e.g., hepatocytes) can be transfected with RNAior siRNA directed against any ABC transporters, SLC transporters or anyother drug metabolizing enzymes to knockdown/knockout the expression ofspecific genes.

In one aspect (1), the disclosure provides a cryopreserved recombinantcell including one or more transiently overexpressed genes encoding aprotein selected from the group consisting of a drug transporter proteinand a drug metabolizing enzyme, or a combination thereof whereinactivity of the drug transporter protein or the drug metabolizing enzymeor combinations is detectable in a population of the cryopreservedrecombinant cell following thaw from cryopreservation.

In an aspect (2), the disclosure provides the invention of aspect (1),wherein said one or more genes encodes a drug metabolizing enzyme.

In an aspect (3), the disclosure provides the invention of aspect (2),wherein the drug metabolizing enzyme is selected from the groupconsisting of cytochrome P450, UDP-glucouronyl transferase, alcoholdehydrogenase, monoamine oxidase and aldehyde oxidase.

In an aspect (4), the disclosure provides the invention of aspect (1),which transiently overexpresses one or more genes encoding a proteinselected from the group consisting of an ATP binding cassettetransporter and a solute carrier transporter protein.

In an aspect (5), the disclosure provides the invention of aspect (4),wherein said one or more genes is selected from the group consisting ofMDR1/Mdr1a/Mdr1b, MRP1/Mrp1, MRP2/Mrp2, MRP3/Mrp3, MRP4/Mrp4, MRP5/Mrp5,MRP6/Mrp6, MRP7/Mrp7, MRP 8/Mrp8, BCRP/Bcrp, BSEP/Bsep, OATP2/Oatp2,OATP1B3/Oatp1b3, OAT1/Oat1, OAT2/Oat2, OAT3/Oat3, OAT4/Oat4, OCT1/Oct1,OCT2/Oct2, OATP1/Oatp1, PEPT1/Pept1, PEPT2/Pept2, OCTN1/Octn1,OCTN2/Octn2, MATE1/Mate1, MATE2K/Mate2, URAT1/Urat1, ASBT/Asbt, andNTCP/Ntcp.

In an aspect (6), the disclosure provides the invention of aspect (4),wherein said one or more genes encodes a protein that is an ATP bindingcassette transporter selected from the group consisting ofMDR1/Mdr1a/Mdr1b, MRP1/Mrp1, MRP2/Mrp2, MRP3/Mrp3, MRP4/Mrp4, MRP5/Mrp5,MRP6/Mrp6, MRP7/Mrp7, MRP 8/Mrp8, BCRP/Bcrp, and BSEP/Bsep.

In an aspect (7), the disclosure provides the invention of aspect (4),wherein said one or more genes encodes a protein that is a solutecarrier transporter selected from the group consisting of OATP2/Oatp2,OATP1B3/Oatp1b3, OAT1/Oat1, OAT2/Oat2, OAT3/Oat3, OAT4/Oat4, OCT1/Oct1,OCT2/Oct2, OCT3/Oct3, OATP1/Oatp1, PEPT1/Pept1, PEPT2/Pept2,OCTN1/Octn1, OCTN2/Octn2, MATE1/Mate1, MATE2K/Mate2, URAT1/Urat1,ASBT/Asbt, and NTCP/Ntcp.

In an aspect (8), the disclosure provides the invention of aspect (4),wherein said one or more genes is selected from OATP1B1*1a, OATP1B1*1b,OATP1B3, OAT1, OAT3, OCT1, OCT2, MATE1 and MATE2K.

In an aspect (9), the disclosure provides the invention of aspect (1),wherein the one or more genes is derived individually from human or ananimal species selected from mouse, rat, guinea pig, dog, and monkey.

In an aspect (10), the disclosure provides the invention of aspect (1),wherein said cell is derived from a mammal.

In an aspect (11), the disclosure provides the invention of aspect (10),wherein said cell is selected from the group consisting of HEK293, CHO,MDCK, LLC-PK1, Caco-2 and V79 cells.

In an aspect (12), the disclosure provides the invention of aspect (10),wherein the mammal is selected from the group consisting of human,monkey, dog, rat, mouse, porcine and hamster.

In an aspect (13), the disclosure provides the invention of aspect (1),wherein said cell comprises a hepatocyte.

In an aspect (14), the disclosure provides the invention of aspect (1),wherein said cell comprises an endothelial cell.

In an aspect (15), the disclosure provides the invention of aspect (1),wherein activity of the protein(s) is detectable in a population of saidcell at least 24 hours post plating following thaw fromcryopreservation.

In an aspect (16), the disclosure provides the invention of aspect (1),wherein activity of the protein(s) is detectable in a population of saidcell at least 48 hours post plating following thaw fromcryopreservation.

In an aspect (17), the disclosure provides the invention of aspect (1),wherein activity of the protein(s) is detectable in a population of saidcell at least 72 hours post plating following thaw fromcryopreservation.

In another aspect (18), the disclosure provides a process of preparingtransiently transfected recombinant cells which transientlyoverexpresses one or more genes encoding a protein selected from a drugtransporter protein and a drug metabolizing enzyme including transientlytransfecting cells with one or more genes encoding a drug transporterprotein or a drug metabolizing enzyme and cryopreserving the transientlytransfected recombinant cells within 48 hrs of transfection.

In an aspect (19), the disclosure provides the invention of aspect (18),wherein the transient transfection step includes electroporation.

Examples

Cells were cultured under standard sterile practices for cell culture,and transiently transfected using EP. Following EP, cells were assayedfor protein activity both before as well as after being frozen, thawedand plated. As detailed below, cells cultured in suspension and adherentcell cultures were both successfully transiently transfected andexhibited substantial activity of the recombinant protein following thawfrom cryopreservation.

Cells Cultured in Suspension

In brief, on Day 1, FreeStyle 293 Cells and 293-F cells were eachpassaged into appropriate sized shaker flasks at a density of0.7-1.0×10⁶ cell/ml using supplemented CD293 medium (i.e., CD293 medium(available from Gibco, Cat. No. 11913-019, Life Technologies Corp.,Carlsbad, Calif.) supplemented with 4 mM L-Glutamine (available fromGibco, Cat. No. 25030-081, Life Technologies Corp., Carlsbad, Calif.))or supplemented Excell™ 293 serum free media (available from Sigma, Cat.No. 14571C, Sigma-Aldrich, St. Louis, Mo.) supplemented with 6 mML-Glutamine. Cell viability and cell number were determined using aCellometer (available from Nexcelom Bioscience, Lawrence, Mass.).

On Day 2, EP of cells was executed. In short, following a determinationof cell viability and cell density, cells were pelleted down by spinningat 100 g for 5 min, after which the media was aspirated and cellsresuspended in 30 ml EP Buffer (available from MaxCyte, Cat. No. B201,MaxCyte Inc., Gaithersburg, Md.). The cell suspension was transferred to50 ml Falcon tubes, pelleted down as described above, and resuspended inan appropriate amount of EP Buffer to reach 100×10⁶ cells/ml which wasused as the cell stock. DNAs to be used for EP were prepared in sterilewater at a final concentration of 5 mg/ml. For each sample, 0.4 ml ofcell stock and DNA was placed in a sterile 1.5 ml eppendorf tuberesulting in a final concentration of 200 μg/ml (Table 4) or 300 μg/mlDNA (Table 10 and Table 11) and cell density of 40×10⁶ cells per sample.

TABLE 4 CELL STOCK SAMPLE CELL VOL. [DNA] # TYPE PLASMID(S) (ml) (ug/ml)A 1, 2 FS293 pOATP1B1 0.4 200 B 3, 4 pCMV6 0.4 200 C  5 EP Buffer (16μl) 0.4 — D 6, 7 293-F pOATP1B1 0.4 200 E 8, 9 pCMV6 0.4 200 F 10 EPBuffer (16 μl) 0.4 —

Samples were transferred into an OC-400 Processing Assembly (availablefrom MaxCyte, Cat. No. OC-400R, MaxCyte Inc., Gaithersburg, Md.) whichfollowed the manufacture instructions for EP of HEK cells. Following EP,the cells were carefully pipetted out and transferred into the bottom ofa 125 ml shaker flask and incubated for 20 min at 37° C. with 8% CO₂,after which pre-warmed 40 ml CD293 media was added into the shaker flaskto reach cell density at 1×10⁶ cells/ml. The cells were incubated for 30min at 37° C. and 8% CO₂. After 30 min recovery, cell viability and celldensity were determined. A portion of cells (i.e., 20×10⁶ cells) wasused for plating and the rest was cryopreserved, or all of the cellswere cryopreserved. It is contemplated that recombinant cells may becryopreserved within 48 hrs of transfection and exhibit activity ofprotein(s) encoded from transfected gene(s) at a detectable levelfollowing thaw from cryopreservation.

For plating cells following EP, 20×10⁶ cells were pelleted down byspinning at 100 g for 5 min and then resuspended in 20 ml pre-warmedplating media (DMEM with high glucose (available from Gibco, Cat. No.11965092, Life Technologies Corp., Carlsbad, Calif.), supplemented with0.1 mM non-essential amino acids (available from Gibco, Cat. No.11140050, Life Technologies Corp., Carlsbad, Calif.), 10% FBS (availablefrom SAFC Biosciences, Cat. No. 12016C, Sigma, St. Louis, Mo.)) (celldensity of 1×10⁶ cells/ml). Cells were placed in 24-well tissue cultureplates poly-D-Lysine coated, Corning Biocoat™ (available from CorningLife Sciences, Tewksbury, Mass.) at a density of 0.2×10⁶ cells/well and0.4×10⁶ cells/well and incubated at 37° C. with 8% CO₂ so as todetermine the impact of seeding density on uptake activity. Media wasreplaced 4 hours later and then every 24 hours until the day ofassaying. On Days 4, cells were assayed for OATP1B1 activity asdescribed below.

For cryopreservation, cells were pelleted then resuspended in freshlyprepared ice-cold freezing media (9 parts supplemented CD293 medium and1 part DMSO which was syringe filtered to sterilize) at a density of10×10⁶ cell/ml. Cryo vials were filled with 1 ml of this cellsuspension, and placed on ice-cold Mr Frosty freezing container(available from Thermal Scientific), which was stored in −80° C. freezerovernight after which the vials were transferred into liquid nitrogen.

Cryopreserved cells were assayed for OATP1B1 activity as describedbelow. In brief, on Day 1, cryopreserved cells were removed from liquidnitrogen to dry ice, and then thawed in a water bath at 37° C. for about2 min. Cells were transferred into 10 ml of plating media as describedabove which is pre-warmed to a temperature of about 37° C. and theviability and cell density determined. Cells were pelleted down andresuspended in supplemented DMEM media at a cell density of 1×10⁶ viablecells/ml. Cells were plated in the same manner described above forplating cells following EP (which had not been cryopreserved) andassayed for OATP1B1 activity at 24, 48 and 72 hrs following platingthereof.

Adherent Cell Cultures

In brief, HEK293 cells were cultured in 5 Layer Corning® CellStack®(available from Corning Inc. Life Sciences, Tewksbury, Mass.) usingplating media containing DMEM (high glucose) available from Gibco Cat.No. 11965118, Life Technologies Corp., Carlsbad, Calif.;Penicillin-Streptomycin (10,000 units/ml) available from Gibco Cat. No.15140-122, Life Technologies Corp., Carlsbad, Calif.; L-Glutamine (200mM) available from Gibco Cat. No. 25030-081, Life Technologies Corp.,Carlsbad, Calif.; Sodium Pyruvate, available from Gibco Cat. No. 11360,Life Technologies Corp., Carlsbad, Calif.; FBS available fromSigma-Aldrich Corp., St. Louis, Mo. in a ratio of 100:1:1:1:10. On Day1, about 24 hrs before EP, HEK293 cells were trypsinized, cell viabilityand cell number determined after which cells were passaged to freshmultilayer chamber flasks at 30-40% confluency. Cells were incubated at37° C. with 5% CO₂.

On Day 2, EP of cells was executed. In short, cells were harvested, cellviability and cell number determined after which cells were pelleteddown by spinning at 100 g for 5 min and the media aspirated. Cells wereresuspended in EP buffer and pelleted down by spinning at 100 g for 5min, then resuspended in an appropriate amount of EP Buffer to reach50×10⁶ cells/ml which was used as the cell stock. DNAs to be used for EPwere prepared in sterile water at a final concentration of 5 mg/ml. Foreach sample used for OC-400 processing assembly, 0.4 ml of cell stockand DNA was placed in a sterile 1.5 ml eppendorf tube resulting in afinal concentration of 50 μg/ml, 100 μg/ml, 200 μg/ml or 400 μg/ml DNAas indicated in FIGS. 5-9 and cell density of 40×10⁶ cells per sample.For each sample used for CL-2 processing assembly, 10 ml of cell stockand DNA was placed in 50 ml sterile conical tube resulting in a finalconcentration of 100 μg/ml DNA.

Samples were transferred into an OC-400 or CL-2 processing assembly(available from MaxCyte, Cat. No. OC-400R and CL2-R, MaxCyte Inc.,Gaithersburg, Md.) which followed the manufacture instructions for EP ofHEK cells. Following EP, the cells were carefully pipetted out andtransferred into 6-well tissue culture plates and incubated for 20 minat 37° C. with 5% CO₂, after which cells were removed and placed in a 50ml conical tube containing pre-warmed plating media. Cell viability andcell density were determined. A portion of cells (i.e., 20×10⁶ cells)was used for plating and the rest was cryopreserved.

For plating cells following EP, cells were pelleted down by spinning at100 g for 5 min and then resuspended in pre-warmed plating media (celldensity of 1×10⁶ cells/ml). Cells were placed in 24-well tissue cultureplates (poly-D-Lysine coated, Corning Biocoat™ (available from CorningLife Sciences, Tewksbury, Mass.)) at a density of 0.4×10⁶ cells/well andincubated at 37° C. with 5% CO₂. Media was replaced 4 hours later andthen every 24 hours until the day of assaying. On Days 4 and 6, cellswere assayed for OATP1B1 activity.

For cryopreservation, cells were pelleted then resuspended in freshlyprepared ice-cold freezing media (9 parts plating medium and 1 part DMSOwhich was syringe filtered to sterilize) at a density of 10×10⁶ cell/ml.Cryo vials were filled with 1 ml of this cell suspension, and placed onice-cold Mr Frosty freezing container (available from ThermalScientific) stored in −80° C. freezer overnight after which the vialswere stored in liquid nitrogen.

Cryopreserved cells were assayed for OATP1B1 activity. Notably, cellswere plated in the same manner described above for plating cellsfollowing EP (which had not been cryopreserved) and assayed for OATP1B1activity (as described below) at 48 hrs following plating thereof.

Assaying Transporter Activity

In brief, substrate solution was prepared for OATP1B1*1a and OATP1B1*1busing 2 μM estradiol-17β-glucuronide (99% of cold E17βG and 1% of[³H]-E17βG); for OATP1B3 using 2 μM CCK-8 (99% of cold CCK-8 and 1% of[³H]-CCK-8); for OAT1 short using 1 μM Para-aminohippurate (PAH) (90% ofcold PAH and 10% of [³H]-PAH); for OAT1 long using 1 μM or 3 μMPara-aminohippurate (PAH) (90% of cold PAH and 10% of [³H]-PAH); forOAT3 using 1 μM or 2 μM Estrone-3-sulfate (99% of cold E3S and 1% of[³H]-E3S); for OCT1 and OCT2 using 30 μM Tetraethylammonium Bromide(100% [¹⁴C]-TEA); for MATE1 and MATE2K using 10 μM Metformin (100%[¹⁴C]-Metformin) or 10 μM Tetraethylammonium Bromide (100% [¹⁴C]-TEA);in Krebs-Henseleit Buffer pH 7.4 (available from Sigma, Cat. No. K3753,Sigma-Aldrich, St. Louis, Mo.) and incubated at 37° C. for at least 20min. Culture media was aspirated from cells to be assayed and cellswashed thrice with pre-warmed KHB Buffer. Cells were subsequentlyincubated with Uptake Buffer at 37° C. for 10 min. For MATE1 and MATE2K,cells were washed and pre-incubated with KHB buffer containing 20 mMNH₄Cl for 10 min. Assays were initiated by adding 0.3 ml substratesolution into each well and incubated at 37° C. for 5 min with samplesfor OCT1 and OCT2 incubated for 10 min.

The reaction was quickly stopped after the incubation period byaspirating substrate solution from cells then washing cells thrice withcold Uptake Buffer. Cells were then incubated with lysing solution (0.1%SDS with 0.1% v/v 1M NaOH in Dulbecco's Phosphate-Buffered Saline (DPBS)buffer) for 15-20 minutes while being shaken. The substrate solution wastriturated and 0.4 ml of the resultant cell lysis placed in 5 mlscintillation tube with 5 ml of scintillation liquid for analysis withscintillation counter.

As illustrated in FIG. 1, cell viability dropped 1-5% after EP relativeto that of the cell stock. Additionally, after cryopreservation, cellviability dropped an additional 10-15% relative to that after EP.Nonetheless, even after EP and thaw from cryopreservation, cellviability is greater than 75%.

Cell morphology and uptake activity was examined followingcryopreservation after 30 min recovery and 24 hrs recovery posttransfection. Table 5 illustrated cell morphology and uptake activitywith 24 hrs recovery was reduced compared to 30 min recovery.

TABLE 5 Recovery Cell time prior to Confluency Uptake Activity SAMPLEcryopreservation at 24 hrs (pmole/mg/min) S:N OATP1B1 24 HOURS 40-50%0.59 0.44 OATP1B1 30 MIN 70-75% 5.78 4.32 VECTOR 30 MIN 90-95% 1.34

Cell morphology and confluency of transfected cells thawed fromcryopreservation were examined after various periods of time followingplating at a density of 0.4×10⁶ cells per well in 24-well poly-D-lysinecoated Corning Biocoat™ plates. In particular, FIG. 2 illustratesOATP1B1 transiently transfected cells cultured at 4 hrs, 24 hrs and 72hrs post plating. Additionally, cell confluency at 24 hrs, 48 hrs and 72hrs post-plating of these cells is recorded in Table 6 below.

TABLE 6 CELLS 24 hrs 48 hrs 72 hrs FS293 with pOATP1B1 80-90%  90-95%80-85% FS293 with pCMV6 vector 70-80%  90-95% 90% 293-F with pOATP1B190-95% 95-100% 80-85% 293-F with pCMV6 vector 90-95% 95-100% 80-85%

Desirably, after EP and cryopreservation, the cells form a monolayer onpoly-D-lysine coated Corning Biocoat™ plates achieving 80-90% confluencyat 24 hrs post-plating, 90%-100% confluency at 48 hrs post-plating.

FIG. 4 illustrates cells, transiently transfected with MATE1, MATE2K,OATP1B3, OAT1 long, OAT1 short, OAT3, and pCMV vector respectively,cultured at 24 hrs post plating after thawed from cryopreservation.

As illustrated in FIG. 5, the expression of Green Fluorescent Protein(GFP) in adhesion HEK293 cells was increased with increasingconcentration of DNA. Additionally, GFP expression increased at the 48hr timepoint relative to the 24 hr timepoint. In particular, GFPtransfection efficiency by EP achieved 100% at 24 hrs with 200 μg/ml DNAand 100% fluorescent cell staining at 48 hrs with 100 g/ml DNA. Hence,GFP protein expression level in transfected cells increased withincreased DNA concentration and at 48 hrs relative to 24 hrs.

Uptake activity of suspension cultured 293 cells transfected withOATP1B1 (pOATP1B1) and control vector (pCMV) were assayed at varioustime points following EP. In brief, transfected cells were plated at adensity of 0.4×10⁶ cells/well in 24-well poly-D-lysine coated CorningBiocoat™ plates following EP or after thaw from cryopreservation.OATP1B1 uptake activity and uptake ratio were determined using probesubstrate, estradiol-17β-glucuronide, in both fresh plated cells(“fresh”) and cryopreserved cells (“cryo”) at various timepoints postplating as detailed in Table 7 below.

TABLE 7 CELLS/ CELL CULTURE PLATING MEDIA, TIME UPTAKE ACTIVITY FRESHPOINT (pmol/mg/min)/confluence UPTAKE OR CRYO (HR) pOATP1B1 pCMV RATIO293-F in 48 15.4 (85%)   0.7 (90%)   22.0 CD293, fresh 293-F in 48 15.1(95-100%) 0.9 (95-100%) 16.8 CD293, cryo FS293 in 24 36.4 1.9 19.2Excell, cryo 48 10.0 0.7 14.3 72 6.6 1.0 6.6 293-F in 24 27.4 1.5 18.3CD293, cryo 48 15.1 0.9 16.8 72 9.9 1.0 9.9 Note: The number appearingin parentheses is the cell confluency at assay time.

OATP1B1 uptake activity and uptake ratio in transfected cells followingthaw from cryopreservation is consistent with those in freshly platedtransfected cells. In both cells types 293-F and FS293, the highestuptake activity and uptake ratio is observed at 24 hrs post plating.

Morphology and cell confluency of transfected cells (FS293 or 293-F)were examined at 24 hrs, 48 hrs and 72 hrs post-plating in 24-wellpoly-D-lysine coated Corning Biocoat™ plates at plating density ofeither 0.4×10⁶ cells/well or 0.2×10⁶ cells/well after thaw fromcryopreservation. Cell confluency at 24 hrs post-plating are summarizedbelow in Table 8. Cell confluency at 48 hrs and 72 hrs are similar tothose achieved at 24 hrs (data not shown). Additionally, FIG. 3 providesimages of transfected cells plated at (A) 0.4×10⁶ cells per well and (B)0.2×10⁶ cells per well 24 hrs post-plating following thaw fromcryopreservation at a confluence of 90-95% and 60-70%, respectively.

TABLE 8 CELLS, CULTURE MEDIA, 0.4 × 10⁶ 0.2 × 10⁶ TRANSFECTED DNACELLS/WELL CELLS/WELL FS293 with pOATP1B1 in Excell 80-90% 30-50% FS293with pCMV vector in Excell 70-80% 50% 293-F with pOATP1B1 in CD29390-95% 60-70% 293-F with pCMV6 vector in CD293 90-95% 80%

For optimal assay performance, plating cells at a density of 0.4×10⁶ ispreferable to that of 0.2×10⁶ as it achieves higher cell confluency andhigher uptake activity.

TABLE 9 UPTAKE ACTIVITY (pmol/mg/min)/confluence UPTAKE CELLS pOATP1B1pCMV6 RATIO FS293 cells, 0.2 × 10⁶ cells/well 10.5 3.0 3.5 FS293 cells,0.4 × 10⁶ cells/well 36.4 1.9 19.2 293-F cells, 0.2 × 10⁶ cells/well20.2 1.5 13.5 293-F cells, 0.4 × 10⁶ cells/well 27.4 1.5 18.3

Following EP, cell viability was examined using trypan blue andhemocytometer or cellometer.

As illustrated in FIG. 6, when using adhesion HEK293 cells, cellviability post EP dropped with increasing amounts of DNA used in EP.Nonetheless, cell viability following transfection with pOATP1B1 wasranged from 89% to 77% and that following transfection with empty vectorwas 90%.

As illustrated in FIG. 7, when using adhesion HEK293 cells, OATP1B1mediated uptake of Estradiol-17β-glucuronide in the fresh platedtransient transfected adhesion HEK293 cells is time-dependent. Notably,uptake activity and uptake ratio increased with increasing amounts ofDNA used in EP. However, OATP1B1 mediated uptake ofEstradiol-17β-glucuronide reduced at the 96 hr timepoint relative to the48 hr timepoint. Further, as illustrated in FIG. 8, the signal to noiseratio (i.e., uptake ratio) of estradiol-17β-glucuronide increased withthe increase of amount of DNA and assay incubation time, in adhesionHEK293 cells transfected with OATP1B1 relative to empty vector at 48 hrspost EP.

As illustrated in FIG. 9, when using adhesion HEK293 cells,estradiol-17β-glucuronide uptake in OATP1B1 transiently expressed HEK293cells using small scale EP device and large scale EP device isconsistent for both uptake activity and signal to noise ratio (i.e.,uptake ratio). 100 μg/ml DNA was used in the experiments.

As illustrated in FIG. 10, when using adhesion HEK293 cells, OATP1B1uptake activity is compared between the cells transfected usingtraditional lipid transfection reagent (control: lipofectamine 2000,available from Invitrogen) and EP using STX, MaxCyte Inc., Gaithersburg,Md. Notably, cells transfected using EP resulted in a pronouncedlygreater signal to noise ratio relative to those cells transfected withlipid transfection reagent.

As illustrated in FIG. 11, when using adhesion HEK293 cells, OATP1B1uptake activity in both freshly plated EP transfected cells and cellsfollowing thaw from cryopreservation was detectable.

Uptake activity of suspension cultured 293 cells transfected withOATP1B1*1a, OATP1B1*1b, OATP1B3, OAT1 long, OAT1 short, OAT3, OCT1,OCT2, MATE1, MATE2K or control vector (pCMV) were assayed at 24 hrs postplating after thaw from cryopreservation. In brief, the transfectedcells were plated at a density of 0.4×10⁶ cells/well in 24-wellpoly-D-lysine coated Corning Biocoat™ plates following EP and after thawfrom cryopreservation. SLC transporter uptake activity and uptake ratiowere determined using probe substrates as indicated at 24 hrs postplating as detailed in Table 10 below.

TABLE 10 UPTAKE ACTIVITY (pmol/mg/min)/ SLC TRANS- trans- UPTAKE PORTERSSUBSTRATE porter pCMV6 RATIO OATP1B1*1a 2 μM E17bG 41.0 1.03 40OATP1B1*1b 2 μM E17bG 32.6 0.88 37 OATP1B3 2 μM CCK-8 28.7 0.2 145OATP1B3 2 μM CCK-8 77.0 0.79 98 OAT1 long 1 μM PAH 13.1 0.3 39 OAT1short 1 μM PAH 9.7 0.3 29 OAT1 long 3 μM PAH 15.0 0.71 21 OAT3 1 μM E3S44.7 1.2 38 OAT3 2 μM E3S 60.9 1.62 38 OCT1 30 μM TEA 127.6 5.63 23 OCT230 μM TEA 100.5 5.53 18 MATE1 10 μM Metformin 71.4 6.0 12 10 μM TEA 46.34.3 11 MATE2K 10 μM Metformin 33.5 5.2 6.5 10 μM TEA 46.6 6.1 7.6

As reflected in Table 10 above, the recombinant cells exhibited stronguptake activity towards their specific prototypical substrate each ofwhich had an uptake ratio above 10. Notably, an uptake ratio above 5indicates a successful process.

As reflected in Table 11, the post-thaw viability for recombinantcryopreserved cells was determined to be above 90%.

TABLE 11 Cells Post-thaw Viability OATP1B1*1a 94.2% OATP1B1*1b 96.1%OATP1B3 95.5% OAT1 long 93.5% OAT3 93.8% OCT1 95.1% OCT2 96.1%

Each of these recombinant cells as well as a control vector (pCMV) wasexamined 24 hrs post-plating (after cryopreservation). Confluency foreach of these cells 24 hrs post-plating was 85% or greater as reflectedin Table 12 below.

TABLE 12 Transfected Cells 24-h confluency OATP1B1*1a 90% OATP1B1*1b 95%OATP1B3 95% OAT1 long 90% OAT3 90% Vector 95% OCT1 95% OCT2 85%

As illustrated in FIG. 12, each of the 8 cryopreserved recombinant cellsformed a confluent monolayer following thawing, plating on Poly-D-Lysineplates and incubation for 24-hrs post-plating.

As illustrated in FIGS. 13-19 and Tables 13-14, the kinetic andinhibition profiles examined in cryopreserved recombinant cellsexpressing a transporter protein was consistent with reported values.Specifically, as illustrated in FIGS. 13A-13C, the kinetics of PAHuptake by recombinant cells expressing OAT1 and inhibition profile ofprobenecid thereof is consistent with reported values. As illustrated inFIGS. 14A-14C, the kinetics of E3S uptake by recombinant cellsexpressing OAT3 and inhibition profile of probenecid thereof isconsistent with reported values. As illustrated in FIGS. 15A-15F, thekinetics of TEA and metformin uptake by recombinant cells expressingOCT1 as well as inhibition profile thereof is consistent with reportedvalues. As illustrated in FIGS. 16A-16E, the kinetics of TEA andmetformin uptake by recombinant cells expressing OCT2 as well asinhibition profile is consistent with reported values. As illustrated inFIGS. 17A-17F, the kinetics of E17βG, E3S and rosuvastatin uptake byrecombinant cells expressing OATP1B1*1a as well as inhibition profile ofE17βG uptake by cyclosporin A is consistent with reported values. Asillustrated in FIGS. 18A-18E, the kinetics of E17βG, E3S androsuvastatin uptake by recombinant cells expressing OATP1B1*1b as wellas inhibition profile of E17βG uptake by cyclosporin A is consistentwith reported values. As illustrated in FIGS. 19A-19E and Tables 13-14,the kinetics of CCK-8, E17βG and rosuvastatin uptake by recombinantcells expressing OATP1B3 as well as inhibition profile of CCK-8 uptakeby cyclosporin A is consistent with reported values.

TABLE 13 SLC Transporter Cells Literature Report K_(m) K_(m) TestTransporter Substrate (μM) (μM) System Literature OATP1B1*1a E17βG 6.26.3 HEK293 P. Sharma, cells et al. Xenobiotica 40: 24. 2010 OATP1B3CCK-8 20.2 16.5 CHO Poirier A, Cells et al., J PharmacokinetPharmacodyn, 2009 OAT1 PAH 87.3 28 HEK293 Ueo H, et al., cells BiochemPharmacol., 2005 OAT3 E3S 4.0 6.3 HEK293 Ueo H, et al., cells BiochemPharmacol., 2005

TABLE 14 Corning ® SLC TransportoCells ™ Literature Report IC50 IC50Test Transporter Substrate Inhibitor (μM) (μM) System LiteratureOATP1B1*1a E17βG Cyclosporin 0.8 0.7 HEK293 M G Soars, et A Cells al.,Drug Metab Dispos, 2012 OATP1B3 CCK-8 Cyclosporin 0.7 0.6 HEK293Bednarczyk D. A Cells Anal Biochem. 2010 OAT1 PAH Probenecid 7.2 6.5 CHOHo ES, et al., J Am Soc Nephrol., 2001 OAT3 E3S Probenecid 8.8 9 S2Takeda M, et al., Eur J Pharmacol., 2001 OCT1 Metformin Cimetidine 230104 HEK293 Sumito I, et al., Cells JPET, 2011 OCT2 Metformin Cimetidine195 124 HEK293 Sumito I, et al., Cells JPET, 2011

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but isintended to cover modifications that are within the spirit and scope ofthe invention, as defined by the appended claims.

What is claimed is:
 1. A cryopreserved recombinant cell comprising one or more transiently overexpressed genes encoding a drug transporter protein, wherein activity of the drug transporter protein is detectable in a population of the cryopreserved recombinant cell following thaw from cryopreservation.
 2. The recombinant cell of claim 1, wherein said one or more genes encode a drug transporter protein selected from the group consisting of an ATP binding cassette transporter and a solute carrier transporter protein.
 3. The recombinant cell of claim 2, wherein said one or more genes is selected from the group consisting of MDR1/Mdr1a/Mdr1b, MRP1/Mrp1, MRP2/Mrp2, MRP3/Mrp3, MRP4/Mrp4, MRP5/Mrp5, MRP6/Mrp6, MRP7/Mrp7, MRP 8/Mrp8, BCRP/Bcrp, BSEP/Bsep, OATP2/Oatp2, OATP1B3/Oatp1b3, OAT1/Oat1, OAT2/Oat2, OAT3/Oat3, OAT4/Oat4, OCT1/Oct1, OCT2/Oct2, OATP1A2/Oatp1a2, OATP2B1/Oatp2b1, PEPT1/Pept1, PEPT2/Pept2, OCTN1/Octn1, OCTN2/Octn2, MATE1/Mate1, MATE2K/Mate2, URAT1/Urat1, ASBT/Asbt, NTCP/Ntcp and combinations thereof.
 4. The recombinant cell of claim 2, wherein said one or more genes is selected from the group consisting of OATP2/Oatp2, OATP1B3/Oatp1b3, OAT1/Oat1, OAT2/Oat2, OAT3/Oat3, OAT4/Oat4, OCT1/Oct1, OCT2/Oct2, OATP1A2/Oatp1a2, OATP2B1/Oatp2b1, PEPT1/Pept1, PEPT2/Pept2, OCTN1/Octn1, OCTN2/Octn2, MATE1/Mate1, MATE2K/Mate2, URAT1/Urat1, ASBT/Asbt, NTCP/Ntcp and combinations thereof.
 5. The recombinant cell of claim 2, wherein said one or more genes is selected from the group consisting of OATP1B1*1a, OATP1B1*1b, OATP1B3, OAT1, OAT3, MATE1, MATE2K and combinations thereof.
 6. The recombinant cell of claim 1, wherein said one or more genes is derived individually from human or an animal species selected from mouse, rat, guinea pig, dog, and monkey.
 7. The recombinant cell of claim 1, wherein said cell is selected from the group consisting of HEK293, CHO, MDCK, LLC-PK1, Caco-2 and V79 cells.
 8. The recombinant cell of claim 1, wherein said cell comprises a hepatocyte.
 9. The recombinant cell of claim 1, wherein said cell comprises an endothelial cell.
 10. The recombinant cell of claim 1, wherein activity of the drug transporter protein is detectable in a population of said cell at least 24 hours post plating following thaw from cryopreservation.
 11. The recombinant cell of claim 1, wherein activity of the drug transporter protein is detectable in a population of said cell at least 48 hours post plating following thaw from cryopreservation.
 12. The recombinant cell of claim 1, wherein activity of the drug transporter protein is detectable in a population of said cell at least 72 hours post plating following thaw from cryopreservation.
 13. A process of preparing transiently transfected recombinant cells which transiently overexpress one or more genes encoding a drug transporter protein, comprising transiently transfecting cells with one or more genes encoding a drug transporter protein and cryopreserving the transiently transfected recombinant cells within 48 hrs of transfection.
 14. The process of claim 13, wherein the transient transfection step comprises electroporation.
 15. The process of claim 13, wherein the transiently transfected recombinant cells are cryopreserved at 30 minutes to 24 hours post transfection.
 16. The process of claim 13, wherein said one or more genes encode a drug transporter protein selected from the group consisting of an ATP binding cassette transporter and a solute carrier transporter protein.
 17. The process of claim 16, wherein said one or more genes is selected from the group consisting of MDR1/Mdr1a/Mdr1b, MRP1/Mrp1, MRP2/Mrp2, MRP3/Mrp3, MRP4/Mrp4, MRP5/Mrp5, MRP6/Mrp6, MRP7/Mrp7, MRP 8/Mrp8, BCRP/Bcrp, BSEP/Bsep, OATP2/Oatp2, OATP1B3/Oatp1b3, OAT1/Oat1, OAT2/Oat2, OAT3/Oat3, OAT4/Oat4, OCT1/Oct1, OCT2/Oct2, OATP1A2/Oatp1a2, OATP2B1/Oatp2b1, PEPT1/Pept1, PEPT2/Pept2, OCTN1/Octn1, OCTN2/Octn2, MATE1/Mate1, MATE2K/Mate2, URAT1/Urat1, ASBT/Asbt, NTCP/Ntcp and combinations thereof.
 18. The process of claim 16, wherein said one or more genes is selected from the group consisting of OATP2/Oatp2, OATP1B3/Oatp1b3, OAT1/Oat1, OAT2/Oat2, OAT3/Oat3, OAT4/Oat4, OCT1/Oct1, OCT2/Oct2, OATP1A2/Oatp1a2, OATP2B1/Oatp2b1, PEPT1/Pept1, PEPT2/Pept2, OCTN1/Octn1, OCTN2/Octn2, MATE1/Mate1, MATE2K/Mate2, URAT1/Urat1, ASBT/Asbt, NTCP/Ntcp, and combinations thereof.
 19. The process of claim 16, wherein said one or more genes is selected from the group consisting of OATP1B1*1a, OATP1B1*1b, OATP1B3, OAT1, OAT3, MATE1, MATE2K and combinations thereof.
 20. A cryopreserved transiently transfected recombinant cell prepared according to the process of claim
 13. 